Microbiome of the freshwater sponge Ephydatia muelleri shares compositional and functional similarities with those of marine sponges

Abstract

Sponges are known for hosting diverse communities of microbial symbionts, but despite persistent interest in the sponge microbiome, most research has targeted marine sponges; freshwater sponges have been the focus of less than a dozen studies. Here, we used 16 S rRNA gene amplicon sequencing and shotgun metagenomics to characterize the microbiome of the freshwater sponge Ephydatia muelleri and identify potential indicators of sponge-microbe mutualism. Using samples collected from the Sooke, Nanaimo, and Cowichan Rivers on Vancouver Island, British Columbia, we show that the E. muelleri microbiome is distinct from the ambient water and adjacent biofilms and is dominated by Sediminibacterium, Comamonas, and unclassified Rhodospirillales. We also observed phylotype-level differences in sponge microbiome taxonomic composition among different rivers. These differences were not reflected in the ambient water, suggesting that other environmental or host-specific factors may drive the observed geographic variation. Shotgun metagenomes and metagenome-assembled genomes further revealed that freshwater sponge-associated bacteria share many genomic similarities with marine sponge microbiota, including an abundance of defense-related proteins (CRISPR, restriction-modification systems, and transposases) and genes for vitamin B12 production. Overall, our results provide foundational information on the composition and function of freshwater sponge-associated microbes, which represent an important yet underappreciated component of the global sponge microbiome.

Fig. 1. Locations for sampling the freshwater sponge Ephydatia muelleri.

Map depicts southern Vancouver Island (British Columbia, Canada). Samples were collected in July 2018 from suitable habitat in the Sooke, Nanaimo, and Cowichan Rivers. Watersheds are shaded blue and sampling locations within each river are indicated in red.

Fig. 2. Alpha and beta diversity across sponges, water, and biofilms.

Boxplots of (a) ASV richness and (b) Shannon diversity calculated via rarefaction and extrapolation, shown for all samples and for each sampling location. Letters represent significant pairwise contrasts (p < 0.05) between sample types (sponge, water, biofilm) within each grouping. Pairwise comparisons were performed using Tukey’s HSD post hoc test. The single biofilm sample from the Cowichan River was not included in any pairwise comparisons, and detailed results for all pairwise comparisons are presented in Table S2. c Aitchison distance-based ordination showing significant differences among sample types. Colored lines show 95% confidence ellipses around sample types, and black dashed lines show 95% confidence ellipses around sponge and water samples separately for each river.

Fig. 3. ASV abundance correlations between sponges and water or biofilms.

ASV abundances in sponges were plotted as a function of ASV abundances in water (left) or biofilms (right). Spearman’s correlation coefficient is given on top of each graph. Colored dots indicate ASVs that were significantly more abundant in sponges (orange), water (blue) or biofilms (green) after Benjamini-Hochberg correction. Selected ASVs are labeled with their lowest taxonomic affiliation. Hash marks along the x- and y-axes indicate ASVs with zero abundance in one sample type but non-zero abundance in the other.

Fig. 4. Microbial composition of sponge, water, and biofilm samples.

Average relative abundances (in percent) of different bacterial phyla based on ASV counts for each sample type at each of sampling location. The two most abundant phyla, Bacteroidetes and Proteobacteria are further subdivided into classes.

Fig. 5. COGs that were differentially abundant between sponge and water samples.

Heat map shows natural log-transformed RPKM values for a subset of the 1417 COGs that were differentially abundant (log₂ fold-change >|1| and Benjamini-Hochberg-adjusted p < 0.05) between sponge (SKE) and water (SKEW) samples. This subset of COGs was chosen because the associated functions are consistently implicated in studies of the marine sponge microbiome (e.g., [27, 57, 58, 71]). COGs are organized by general functional categories.

Fig. 6. Genomic composition of metagenome-assembled genomes.

Relative abundances of each metagenome-assembled genome (MAG) in sponge and water samples are shown on the left. On the right, the number of copies of each COG in each MAG is shown for select groups of COGs that were differentially abundant between sponge and water samples (see Fig. 5). Abbreviations: Str. m., structural motifs; R/M, restriction/modification.